Nerve repair unit and method of producing it

ABSTRACT

A nerve repair unit comprising a resorbable polymeric support and an alginate matrix containing human Schwann cells is enclosed. A method of producing the nerve repair unit is also described. The Schwann cells are preferably cells cultured from a nerve biopsy sample from the patient who is going to receive the nerve repair unit as an implant.

The present invention relates to a nerve repair unit and a method ofproducing it. The nerve repair unit comprises a resorbable polymericsupport and an alginate matrix containing human Schwann cells.

BACKGROUND

It is well known that neural cells undergo changes after nerve injury,and some cells die. Further, it is known today that this cell death isextensive and results in changes of the projection pattern that theinjured nerve has on the spinal cord level. Despite optimalmicrosurgical repair immediately after an induced experimental animalnerve injury a 25-50% loss of nerve cells arises, and is accompanied byan even greater loss of sensory reflex contacts inside the spinal cord(1-8). This nerve cell death is initiated within the first few daysafter the nerve injury and continues for several months (9). This celldeath contributes to the impaired recovery of sensation in patients withnerve injuries, since a loss of nerve cells makes it more difficult foroutgrowth of sensory nerves to the target area (10).

For reduction of this cell death, groups of small proteins (growthfactors) are used, which are of great importance for the nerve cellfunction and survival. At nerve injury, these factors are released fromcells, so-called Swann cells, that surround the injured nerve ends.However, due to leakage to the surrounding tissue, the concentrationdoes not become high enough to prevent the nerve cell death. Byintroducing these factors into the spinal cord canal during the nervehealing, it has been demonstrated on rat that they can completelyeliminate the nerve cell death (11).

In clinical practice most patients with major proximal nerve injuries(so-called plexus injuries) after difficult childbirth and trafficaccidents are subjected to surgery after 4-12 weeks, since this time isnecessary for evaluating the extent of the nerve injuries.

It would be desirable, especially in these emergency cases, when thepatients come to the hospital, to take a microscopic nerve biopsy forcultivation of the patient's own Schwann cells. At the time forreconstruction of the injured area, it would be desirable to have meansfor substituting lost nerve tissue with the patient's own Schwann cells.

DESCRIPTION OF THE INVENTION

The present invention provides means for repair of injured human nerves.Thus, the invention is directed to a nerve repair unit comprising aresorbable polymeric support and an alginate matrix containing humanSchwann cells.

The resorbable polymeric support may be any commercially availablesupport or support described in the literature suitable forimplantation, especially a resorbable conduit for nerve regeneration.

In an embodiment of the invention the polymer of the resorbablepolymeric support is selected from the group consisting ofpolyhydroxybutyric acid, polyglycolic acid and polylactic acid. In apresently preferred embodiment the polymer is polyhydroxybutyric acid(PHB).

In a presently most preferred embodiment the resorbable polymericsupport is a polyhydroxybutyric acid conduit.

The choice of alginate as the matrix to be used in the present inventionwas preceded by experimental work evaluation also Collagen Type I(Sigma: C7661); Fibrin glue (Tisseel™, Immuno); Hyaluronic acid (HylanG-F 20, Biomatrix) and Matrigel®: growth factor reduced Matrigel(Collaborative Biomedical Products, Becton Dickinson Labware). They wereall inferior to alginate for the purpose of the present invention.

In an embodiment of the invention the alginate matrix is an ultrapure,low viscosity mannuronic acid alginate.

The human Schwann cells may be received from a culture of a biopsysample from e.g. a branch of the sural nerve of the leg of a donor.Preferably the donor is the patient who is to receive the nerve repairunit of the invention. In case the patient has an open injury, a nervebiopsy sample may be taken from the damaged peripheral nerve forculturing of the Schwann cells.

Another aspect of the invention is directed to a method of producing anerve repair unit comprising a resorbable polymeric support and analginate matrix containing human Schwann cells. The method comprises thesteps of mixing an alginate in isotonic saline solution with a humanSchwann cell suspension in fibronectin, supplying the mixture to theresorbable polymeric support, setting the alginate matrix with asolution of calcium chloride in human cell culture medium, rinsing withthe human cell culture medium and keeping the nerve repair unit in thehuman cell culture medium until use.

In an embodiment of the method of the invention the polymer of theresorbable polymeric support is selected from the group consisting ofpolyhydroxybutyric acid, polyglycolic acid and polylactic acid, thealginate is ultrapure, low viscosity mannuronic acid alginate and thehuman cell culture medium is Dublbecco's Minimum Eagles Medium plusGlutamax® (DMEM).

In another embodiment of the method of the invention the human Schwanncells are produced by transporting a sample of a peripheral nerve of ahuman patient in a transfer medium at ambient temperature within 24hours to a competent laboratory, upon arrival washing the sample with ahuman culture medium, removing the perineurum, dividing the nervebranches into fascicles and cutting them into pieces, washing the pieceswith a human culture medium and placing them into a culture flask coatedwith both poly-D-lysine and human laminin and covering them with aSchwann cell culture medium, followed by incubation at 37° C. for up to10 days, changing the medium every 2 days, and removing the nervesegments for digestion in a container with cell culture medium pluscollagenase I and dispase I until the segments have broken down,followed by trituration, filtration, washing and centrifugation,resuspending the resulting pellet in transfer medium and plating thesuspension on a culture flask coated with both poly-D-lysine and humanlaminin and after 24 hours changing the medium to a Schwann cell culturemedium, followed by cultivation for at least 7 days under conditionsremoving possible fibroblast contamination and changes of culture.medium until the Schwann cells are confluent and ready to split,aspirating the medium and suspending the cells in a trypsinized solvent,followed by centrifugation and washing of the pellet, counting theSchwann cells with a haematocytometer and plating them on a cultureflask coated with both poly-D-lysine and human laminin and covering themwith a Schwann cell culture medium at a density of 5×10⁵ cells/25 cm²,culturing the cells for at least 14 days under change of medium everyday to produce an appropriate cell number for transplantation to thepatient.

In the experimental part of this description it is shown, in an animalmodel, that allogenic cells have a survival time that is shortercompared to syngenic cells in vivo when no immunosuppression is used.However, allogenic Schwann cells have a survival time, prior torejection, that may be sufficient for minor nerve injuries with shorterdefects in the nerve tissue.

The invention will now be illustrated by description of experiments, butit should be understood that the scope of protection is not limited tospecifically mentioned details.

Experiments

Materials Used in the Experiments Resolvable polymer Poly hydroxybutyrate (PHB) sheets Astra-tech, Sweden Matrix Alginate, low viscositymannuronate (LVM) Pronova Biomedical, Norway Batch no: 210-241-02Calcium chloride Sigma Fibronectin, 0.1%, bovine plasma Sigma Tissueculture Cell growth medium DMEM plus: 10% fetal calf serum andPenicillin/Streptomycin (100 iu-100 μg) Collagenase I WorthingtonBiochemicals, Code; CLS-I, 125 U/mg. 1% working solution made with DMEMand aliquoted in 300 μl and kept at −20° C. Complement, rabbit antiCedarlane Labs, Cat No: CL3051, 2 vials mouse reconstituted at a time,kept on ice at all times, 1 ml sterile water added to each vial,filtered through 0.45 μM Filter, stored in liquid Nitrogen as 250 μlaliquots. Cytosine-β-D- Sigma, Cat No: C3639/C8779, Stock solution ˜1mM: arabinofuranoside 5′-tri- 2 mg dissolved in 4 ml PBS, filtered 0.2μm and Phosphate (Arac-C) stored at −20° C., 250 μl aliquots Dispase I:Roche, Cat No: 210 455, 5 mg. Stock solution: 1 ml of sterile wateradded, stored at −20° C. as 0.1 ml aliquots, i.e. each aliquot ˜3 U.DMEM with HEPES Gibco, Cat No: 22320 DMEM, high glucose (4.5 g/l Gibco,Cat No glucose) Dulbecco's Modified Eagle's Gibco, Cat No: 21885 Mediumplus Glutamax (DMEM) Fetal Calf Serum (FCS) Imperial Laboratories, 500ml, Batch No: 53265, Heat deactivated at 56° C. for 35 minutes, storedat −20° C. Forskolin Calbiochem, Cat No: 344270, 5 mM stock solutionmade by dissolving 2.0525 mg/ml in DMSO. Stored at −20° C. 50/100 μlaliquots BMX, Isobutyl-1- Sigma, Cat No: I-5879, Stock solution 50 mM:FW: Methylxanthine 222.2, 100 mg dissolved in DMSO. Stored at −20° C. as200 μl aliquots. Insulin, Bovine Pancreas, Sigma, Cat No: I 1882, Lot:57H4626, Stock solution culture tested 10 mg/ml; 10 ml of acidifiedWater (150 μl of glacial acetic acid added to 15 ml of distilled water,in fume cupboard, 0.2 μm filtered). Dissolved slowly re- filtered with0.8/0.2 μm. Stored at 4° C. as I ml aliquots. Mouse Anti Human Thy 1.1Serotec, Cat NO: MCAP90, 2 ml, IgG1 Mouse Anti Mouse/Rat Serotec, CatNo: MCA04G, 0.25 mg/0.25 ml, IgM Thy 1.1 Penicillin/Streptomycin Gibco,Cat: 15070-022, Working concentration: 100 iu-100 μg, by adding 2 ml to100 ml of DMEM. Poly-D-Lysine, Lyophilised, Sigma, Cat No: P7280 MW:30,000-70,000. Schwann cell growth Rat Schwann cell growth medium (madewith high, medium (human) 4.5 g/l, glucose DMEM plus 0.5 mM IBMX, 1.1 mlof stock and 2.5 μg/ml insulin, 28 μl for 100 ml. Schwann cell growthCell growth medium plus forskolin, 5 μM, GGF 126 ng/ml medium (neonatalrat) (of Batch rhGGF2 121195): To 100 ml of cell growth medium 112 μl offroskolin stock 3 μl of GGF added. Trypsin/EDTA 0.05%/0.02% Gibco, Cat:45300-019Human Schwann Cell CultureNerve Collection

Peripheral nerve samples are taken from small nerve branches from thesural nerve of the leg. Under sterile conditions, the samples areimmediately collected in transfer medium (OptiMEM) and kept at roomtemperature for no longer than 24 hours while transferred to thelaboratory. On arrival, the nerve samples are washed twice with DMEM.

Using an operating microscope, the perineurium is removed and the nervebranches are divided into fascicles and cut into 1 mm pieces. The piecesare washed with DMEM/HEPES, put in a 25 cm² culture flask and coveredwith minimal volume human SC growth medium such that the segmentsremained attached to the flask rather than floating. The culture flaskhas to be coated with both poly-D-lysine and human laminin. Nerve piecesare incubated with SC culture medium at 37° C. for 10 days, with mediumchanges every 2 days. At the end of the incubation period, the segmentsare gently removed.

Digestion and Purification

Day 1 The pre-digestion incubation encourage fibroblast migration out ofthe nerve segments as well as SC mitosis within the nerve. The nervesegments are then digested in a universal container with 2 ml of cellculture medium plus 125 U/ml of collagenase I and 0.8 U/ml of dispase I.The container is kept at 37° C. in a flask shaker to allow gentle mixingfor 2 hours. If the pieces have not broken down, thus not yet digested,the process is continued for further 30-60 minutes. The resultantmixture is triturated through decreasing calibre needles (19G, 21G and23G) filtered through a 70 μm cell filter and the filter is flushed with5 ml of DMEM. The cell suspension is centrifuged at 800 rpm and thepellet re-suspended in 10 ml of OptiMEM and plated on a PDL/laminindouble coated flask. After 24 hours, most SC have settled and the mediumis changed to a human SC growth medium.

Unlike rat SC, human SC cultures do not routinely undergoimmuno-purification as the pre-digestion incubation eliminate most ofthe fibroblasts. However, anti human Thy 1.1 antibodies together withrabbit complement, are sometimes used to eliminate excess fibroblastgrowth.

Day 2 The culture flasks are checked for cell attachment and infection.The medium is carefully taken off and the cells are gently washed withDMEM/HEPES. After washing, 5 ml of growth medium containing 10 μMcytotoxic agent cytosine-β-D-arabinofiuranoside (Arac C) is added andthe flasks are returned to the incubator.

Day 3 The cultures are checked for fibroblast overgrowth and incubationwith fresh medium containing with Ara-C is repeated.

Day 4 The old medium is aspirated, followed by 4 gentle washes of cellswith DMEM/HEPES. 5 ml of SC growth medium is added to the culture andthe flasks are incubated until the SC are confluent, which takes up to 4days.

Day 5-8: Fibroblast depletion: This stage is undertaken once SC (mixedwith fibroblasts) are sub-confluent. SC are lifted off the flask byadding 0.25% trypsinl EDTA (2 ml for a 25 cm² flask, 3-4 ml for a 75 cm²flask). After approximately 5 minutes incubation the cells start to liftoff, then the flask is sharply tapped to detach completely all thecells. Following a check under the inverted microscope to ensurecomplete cell detachment, 5 ml of cell growth medium is added to stopthe trypsin action. The suspension is centrifuged at 800 rmp for 5minutes. The supernatant is aspirated and the cells are washed again andcentrifuged leaving a residual of around 0.1 ml. The cells arere-suspended and 500 μl of diluted mouse anti rat Thy 1.1 antibody (dil.1:1000 in DMEM) is added and the mixture is incubated for 10 minutes.250 μl of complement is added and the cell suspension is incubated forfurther 30 minutes, with occasional mixing. 10 ml of cell growth mediumis added to the cell suspension and centrifuged at 800 rmp for 5minutes. The cell pellet is re-suspended in 5 ml of SC growth medium andincubated in a PDL/laminin coated 25 cm² flask. This procedureeliminates most of the fibroblast contamination. However, on occasionthe procedure is repeated if fibroblast contamination became a problemwhen SC are passaged. Contamination could easily be detected due to thedifferent morphology of the cells. SC are bipolar cells with longprocesses, while fibroblasts show larger cell bodies with shortprocesses.

When the SC are confluent and ready to split, they assume acharacteristic swirl pattern. The medium is aspirated and the cells arelifted off by addition of 0.25% trypsin EDTA. The suspension istransferred to a universal container and centrifuged at 800 rmp for 5minutes.

Culturing for Transplantation

The SC are washed with DMEM/HEPES, counted with a haemocytometer andplated on a PDL/laminin double coated flask with SC growth medium atdensity of 5×10⁵ cells/25 cm².

Human SC grow more slowly than neonatal rat SC and need medium changeevery day.

To achieve an appropriate cell number for transplantation culturing hasto continue for >2 weeks after purification.

Neonatal Schwann Cell Culture

Sciatic nerves from 20 neonatal Lewis rats were harvested and digestedwith 1% collagenase I (Worthington Biochemicals) and 0.25% trypsin(Gibco) over 45 minutes. The digestant was triturated through 21G and23G needles and filtered through a 70 μm cell filter (Falcon). Thefilterant was then centrifuged at 800 rpm for 5 minutes and cells wereresuspended in basic medium: Dulbecco's Minimum Eagles Medium plusGlutamax®, DMEM (Gibco), penicillin, 100 iu/ml and streptomycin 100g/ml, (Gibco), 10% fetal calf serum, PCS (Imperial Laboratories) andplated on a 25 cm² poly-D-lysine (Sigma) coated flask and kept at 37°C., 95% humidity, 5% CO₂. The following day the medium was changed tobasic medium containing 10 μM cytosine-β-D-arabinofuranoside (Sigma) andincubated for 38 hours to stop fibroblast growth. The majority of thecells remaining were SC and the medium was changed to SC growth mediumi.e. basic medium with the addition of recombinant glial growth factorII 63 ng/ml (Max^(1/2) activity, 4.8 μg/ml) (Cambridge NeuroScience),and 10 μM forskolin (Calbiochem). Once the cells became confluent thefinal stage of purification was carried out, cells were trypsinised(Gibco) and the suspension was centrifuged at 800 rpm for 5 minutes.Cells were resuspended in 1 ml of medium containing mouse anti Thy 1(di. 1:1000, Serotec, MCA04) incubated at 37° C. for 30 minutes,followed by addition of 250 μl of rabbit anti mouse complement(Cedarlane) and further incubation of 15 minutes. The cells (around120×10⁵) were then washed and grown on poly-D-lysine (Sigma) coatedflasks, the medium was changed every 48 hours and SC were split, 1 in 3,on confluence (12, 13). At the second passage, following purification,the rats SC were transduced.

Genetic Labelling of Schwann Cells

The retroviral vector pMFG lacZnls (14) was utilised to introducemodified E. coli lacZ marker gene that encodes the β-galactosidaseprotein and nuclear localisation sequence (nls) into SC. For thispurpose the Moloney Murine Leukaemia Virus (MMLV) packaging cell linePT67 (Clontech, USA) was used. This contains the three retroviralstructural genes in its genome but lacks the packaging signal which wasprovided in trans as part of the MFG lacZ nls containing the signal andtwo long terminal repeats (LTR) with lacZ gene cloned between them. Inthis way retrovirus encoding the lacZ gene could transduce SC, but thetransduced SC could not produce any further retrovirus due to theabsence of the retroviral structural genes from their genome. A stablePT67 lacZ nls producing clone (titre approximately 10⁶/ml ) was isolatedand used throughout this study.

Ten ml of growth medium (high glucose DMEM/FCS) was put on a confluentlayer of PT67 lacZ nls producers and kept overnight at 32° C. Afterincubation the medium containing retroviral particles was filteredthrough a 0.45 μm filter (Nalgene) and added to a flask of 70% confluentSC together with the polyanion polybrene (Sigma) at 8 μg/ml. Thetransducing medium was left on SC for 4 hours at 32° C., the medium wasthen taken off and SC growth medium added (15). The SC culture flask waskept at 37° C. overnight and this cycle was repeated three timesconsecutively over 3 days. Transduction rate was 83% prior totransplantation as assessed by 5-bromo-4-chloror-3-indoyl-β-Dgalactosidase (X-gal) staining to detect β-galactosidase expression(16).

Implant Preparation

Schwann Cell Suspension

Transduced SC were expanded in culture (no more than 7 passages) and astock of concentrated SC suspension (160×10⁶/ml) in fibronectin (Sigma)was prepared and kept at 4° C. prior to use, for no longer than 30minutes.

Alginate Matrix and Conduit Filling

A sterile 4% stock solution of ultrapure, low viscosity mannuronic acidalginate (Pronova, Norway) in 0.9% saline solution was prepared. Finalmatrix was prepared by mixing the stock alginate (50:50 v/v) withfibronectin or SC suspension in fibronectin in order to obtain a final80×10⁶/ml concentration of SC. PHB conduits (Astra Tech, Sweden) werecarefully filled with 30 μl of alginate matrix and alginate was set in0.1M CaCl₂ for 2 minutes and filled, conduits were gently rinsed twicein DMEM. Conduits were kept in DMEM at 4° C. for no longer than 2 hoursprior to implantation.

Nerve Repair Model

Using an operating microscope (Zeiss, Germany), PHB conduits containingSC from Lewis rat origin were grafted in adult male Lewis (forsyngeneic) or Dark Agouti (for allogencic) rats (Harlan), average weightof 180 g, to bridge a gap of 1 cm in the left sciatic nerve. These twostrains of rats have differences in their major histocompatibilitycomplexes (MHC) (17). Conduits without SC were also implanted in aseparate group of animals and served as control. Animals were sacrificedas 2, 3 or 6 weeks (n=6 each group) and the conduits were harvested,fixed in Zarnboni's fixative and then rinsed in 0.01 M PBS containing15% (w/v) sucrose and 0.1% (w/v) sodium azide and kept at 4° C.

Tissue Process and Analyses

The specimens were blocked in OCT compound (Tissue-tek, Sakura, Japan),placing a piece of rat liver next to the proximal end of the nerve toidentify the orientation of each sample. The specimens were sectionedlongitudinally, 15 μm thick, using a cryostat (Bright). Chemical X-galstaining and then fluorescent immunohistochemistry were used to examinelabelled SC and regeneration and immunological parameters.

Axonal Regeneration and Schwann Cell Ingrowth

Axonal regeneration distance, the amount of new axonal growth as well asthat of SC ingrowth into the conduits were quantified after doubleimmunostaining on the same section. After blocking with 1% normal ratand goat sera, the sections were stained using a combination of antiserato S100 for SC identification. (dill: 1:000, rabbit polyclonal, Dako)and pan-neurofilaments (PanNF) for regenerating axons (dil 1:1000, mousemonoclonal, Affinit). Sections were incubated for 2 hours at roomtemperature, then washed twice with PBS (5 minutes each wash). Thesecondary fluorescence conjugated antibodies (dil, 1:100, goatanti-rabbit FITC conjugated and goat anti-mouse Cy3 conjugated) wereadded and the sections incubated for another 1 hour at room temperature.The sections were then washed with PBS (3×5 min.) and mounted withVectorshield fluorescent mountant (Vector Labs Inc,). Axonalregeneration distance was measured from the proximal stump into thegraft, using a calibrated microscope graticule (mean of 3non-consecutive sections per animal). The area, of immunostaining wastaken as a measure of the quantity or the axonal regeneration. and SCingrowth into the conduits and was measured across a fixed point toallow comparison between groups, at 3 mm the proximal edge of theconduits and expressed as the percentage of immunostaining per field. Aband of images were taken across the whole of the conduit and theoverlapping images edited to avoid duplicate measurement. The imageswere captured with a SPOT digital camera (Diagnostic Images Inc) andanalysed using a PC based image analysis software (Image Pro Plus,version 4, Media Cybernetics, USA).

Transplanted Schwann Cell

X-gal staining was performed on the sections to assess the presence oftransduced SC. Sections were examined and scored semi-quantitatively ashigh, medium, low or zero. In order to assess the involvement oftransplanted SC in the regeneration process, chemical X-gal staining wascarried out on the same section as immunostaining for PanNF and S100, asabove and the sections were examined using a combination of transmittedlight and epifluorescent microscopy.

Schwann Cell Characterisation

After blocking with 1% normal rat and goat sera, the following antiserawere used for immunostaining on separate but consecutive sections:myelin basic protein (MBP, marker of myelinating SC, mouse monoclonaldil, 1:500, Boehringer), p75 (marker of de-differentiated SC mousemonoclonal dil. 1:40, Boehringer) and neural cell adhesion molecule(NCAM, marker of un-myelinating SC rabbit polyclonal, dil. 1:700,Chemicon). Sections were incubated at 4° C. overnight and, then washedtwice with PBS (5 minutes each). The secondary fluorescence conjugatedantibodies (dil. 1:100, goat anti-rabbit FITC conjugated and goatanti-mouse Cy3 conjugated) were added and the sections incubated foranother 1 hour at room temperature. The sections were then washed withPBS (3×5 min.) and mounted with Vectorshield fluorescent mountant(Vector Labs Inc.). The percentage area of immunostaining in a fixedarea was taken as a measure of expression of each SC phenotypic markers.For each section, a digital image was taken at a fixed point, 3 mm fromthe proximal nerve end and equidistant from the walls of conduit. Thepercentage area of immunostaining was measured for each field using a PCbased image analysis software as above section (mean of 3non-consecutive sections per animal and per staining).

Quantification of MHC Class I and II

Immunostaining was carried out as above, with overnight incubation ofthe sections using the following antisera. MHC I (mouse monoclonal dil1:20 Serotec, MCASIG, clone OX-18); MHC II (mouse monoclonal dil 1:20Serotec, MCA46A anti]-A, clone MRC OX-6). Following immunostainingsections were examined without delay and the intensity of fluorescencewas taken as measure of the level of expression of MHC I and II (2random fields were examined per section and 3 non-consecutive sectionsper animal and-per staining). Digital images were taken at fixedsettings to give comparable intensity measurements. Intensity of thefield was measured using Image Pro Plus software, the calibration curvewas a default straight line setting, which was confirmed withfluorescent beads of standardised luminescence.

Lymphocyte and Macrophage Count

In order to assess the immune response to the conduits the number ofB-lymphocytes, T-lymphocytes and macrophages were counted followingimmunostaining. The following antisera were used with overnightincubation at 4° C.: CD2 (for B-lumphocytes, mouse monoclonal dil, 1:30Serotec), CD45R (for T-lymphocytes mouse monoclonal dil. 1:60, Serotec)and macrophage (mouse monoclonal dil. 1:200, Serotec). Immunostainingfor each antibodies was done on separate but consecutive, sections toallow comparison between the different primary antisera (3 sections persample and antibody). The number of positively stained cells weremanually counted across the whole conduits and expressed as the meannumber of stained cells per section.

Statistical Analysis

One way analysis of variance (ANOVA) was performed to assess significantdifference between groups, Tukey's test for comparisons betweenexperimental and control groups, using a SigmaStat statistical analysispackage (Jandel Corp, USA).

Summary of Results

An identifiable and pure population of cultured Schwann cells (SC) wasobtained. Transduction of lacZ genetic label was carried out and astable population of genetically modified SC was obtained. Transduced SCproperties and lacZ expression were preserved in vitro for 6 months ofcontinuous culture.

Suspension matrix is required for SC transplantation and the suitabilityof alginate hydrogel was confirmed by in vitro tests to support SCproliferation and neurite sprouting in a neuron-glial co-culture.Defects in the rat sciatic nerve injury was bridged using resorbableconduits containing SC. The results showed that the optimal number of SCrequired to enhance axonal regeneration was 80×10⁶/ml and alginatetogether with SC further improved regeneration. Followingtransplantation of syngeneic and allogeneic SC both improved axonalregeneration distance, but the quantity of regeneration was better andmore sustained with syngeneic SC.

References:

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1. Nerve repair unit comprising a resorbable polymeric support and analginate matrix containing human Schwann cells.
 2. Nerve repair unitaccording to claim 1, wherein the polymer of the resorbable polymericsupport is selected from the group consisting of polyhydroxybutyricacid, polyglycolic acid and polylactic acid.
 3. Nerve repair unitaccording to claim 1, wherein the alginate matrix is an ultrapure, lowviscosity mannuronic acid alginate.
 4. Nerve repair unit according toclaim 1, wherein the resorbable polymeric support is apolyhydroxybutyric acid conduit.
 5. Method of producing a nerve repairunit comprising a resorbable polymeric support and an alginate matrixcontaining human Schwann cells, comprising the steps of mixing analginate in isotonic saline solution with a human Schwann cellsuspension in fibronectin, supplying the mixture to the resorbablepolymeric support, setting the alginate matrix with a solution ofcalcium chloride in human cell culture medium, rinsing with the humancell culture medium and keeping the nerve repair unit in the human cellculture medium until use.
 6. Method of producing a nerve repair unitaccording to claim 5, wherein the polymer of the resorbable polymericsupport is selected from the group consisting of polyhydroxybutyricacid, polyglycolic acid and polylactic acid, the alginate is ultrapure,low viscosity mannuronic acid alginate and the human cell culture mediumis Dublbecco's Minimum Eagles Medium plus Glutamax® (DMEM).
 7. Method ofproducing a nerve repair unit according to claim 5, wherein the humanSchwann cells are produced by transporting a sample of a peripheralnerve of a human patient in a transfer medium at ambient temperaturewithin 24 hours to a competent laboratory, upon arrival washing thesample with a human culture medium, removing the perineurum, dividingthe nerve branches into fascicles and cutting them into pieces, washingthe pieces with a human culture medium and placing them into a cultureflask coated with both poly-D-lysine and human laminin and covering themwith a Schwann cell culture medium, followed by incubation at 37° C. forup to 10 days, changing the medium every 2 days, and removing the nervesegments for digestion in a container with cell culture medium pluscollagenase I and dispase I until the segments have broken down,followed by trituration, filtration, washing and centrifugation,resuspending the resulting pellet in transfer medium and plating thesuspension on a culture flask coated with both poly-D-lysine and humanlaminin and after 24 hours changing the medium to a Schwann cell culturemedium, followed by cultivation for at least 7 days under conditionsremoving possible fibroblast contamination and changes of culture mediumuntil the Schwann cells are confluent and ready to split, aspirating themedium and suspending the cells in a trypsinized solvent, followed bycentrifugation and washing of the pellet, counting the Schwann cellswith a haematocytometer and plating them on a culture flask coated withboth poly-D-lysine and human laminin and covering them with a Schwanncell culture medium at a density of 5×10⁵ cells/25 cm², culturing thecells for at least 14 days under change of medium every day to producean appropriate cell number for transplantation to the patient.